Sterols are known to play at least two critical roles in plants: as bulk components of membranes regulating stability and permeability (Bach et al. (1997) Prog. Lipid Res. 36:197–226) and as precursors of growth-promoting brassinosteroids (BRs; Fujioka and Sakurai (1997) Nat. Prod. Rep. 14:1–10). Lesions in brassinosteroid (BR) biosynthetic genes result in characteristic dwarf phenotypes in plants. Understanding the regulation of BR biosynthesis demands continued isolation and characterization of mutants corresponding to the genes involved in BR biosynthesis.
Sterol biosynthesis in plants has been studied extensively through enzyme purification or gene cloning (Grunwald (1975) Annu. Rev. Plant Physiol. 26:209–236; Goodwin (1979) Annu. Rev. Plant Physiol. 30:369–404; Benveniste (1986) Annu. Rev. Plant Physiol. 37:275–308; Bach and Benveniste (1997) Prog. Lipid Res. 36:197–226). FIG. 1 shows the proposed biosynthetic pathway from squalene to brassinolide (BL). A major difference between photosynthetic and nonphotosynthetic organisms is that cyclization of squalene 2,3-oxide is bifurcated to a different route for each system (Benveniste (1986) Annu. Rev. Plant Physiol. 37:275–308). In animals and yeast, squalene 2,3-oxide is cyclized to lanosterol, whereas in photosynthetic organisms it is cyclized to cycloartenol (Nes and McKean (1977) Biochemistry of Steroids and Other Isopentenoids. (Baltimore, Md.: University Park Press)). Accordingly, photosynthetic organisms require somewhat different biosynthetic enzymes, such as cycloartenol synthase (Corey et al. (1993) Proc. Natl. Acad. Sci. USA 90:11628–11632) and cycloeucalenol-obtusifoliol isomerase, which are required to open the cyclopropane ring in cycloartenol (FIG. 1). However, most of the enzymatic steps are shared between the two different pathways.
In plants, sterols are subject to a series of modifications before conversion to BL. Different sterols, such as 24-methylenecholesterol (24-MC), campesterol (CR), isofucosterol, and sitosterol, are converted to the BL congeners dolicholide, BL, 28-homodolicholide, and 28-homoBL, respectively, in a species-specific manner (Fujioka et al. (1997) Plant Cell 9:1951–1962; Sasse (1997) Physiol. Plant. 100:696–701). The BR-specific pathway diverges into the early and the late C-6 oxidation pathways. In the early C-6 oxidation pathway, introduction of a 6-oxo group occurs before the vicinal hydroxylation reactions at the side chain, whereas it occurs after these hydroxylations in the late C-6 oxidation pathway (FIG. 1; Choi et al. (1997) Phytochemistry 44:609–613).
Several mutants, such as constitutive photomorphogenesis and dwarfism (cpd), deetiolated2 (det2), and dwarf4 (dwf4), have been shown to be defective in the BR-specific pathway (Li et al. (1996) Science 272:398–401; Li et al. (1997) Proc. Natl. Acad. Sci. USA 94:3554–3559; Szekeres et al. (1996) Cell 85:171–182; Choe et al. (1998) Plant Cell 10:231–243). These BR biosynthetic dwarfs share a characteristic dwarf phenotype, which includes short robust stems, reduced fertility, prolonged life cycle, and dark-green, round, and curled leaves when grown in the light. In the dark, these mutants exhibit short hypocotyls and expanded cotyledons. cpd (dwf3) mutants are only rescued by 23α-hydroxylated compounds (Szekeres et al. (1996) Cell 85:171–182). The CPD gene was shown to encode a cytochrome P450 steroid hydroxylating enzyme (CYP90A1). In addition, Li et al. (1996) Science 272:398–401 and Li et al. (1997) Proc. Natl. Acad. Sci. USA 94:3554–3559 showed that det2/dwf6 is blocked in the C-5 reduction step. DET2 was found to be homologous to steroid 5α-reductases. Like its animal equivalents, DET2 successfully converted progesterone (3-oxo-Δ4,5 steroid) to 4,5-dihydroprogesterone in a human cell line. In addition, the human 5α-reductase gene effectively complemented det2 mutants (Li et al. (1997) Proc. Natl. Acad. Sci. USA 94:3554–3559). Recently, it has been shown that DWF4 encodes a cytochrome P450 whose amino acid sequence is 43% identical to CPD; DWF4 has been named CYP90B1 (Choe et al. (1998) Plant Cell 10:231–243). Based on results from feeding studies using BR biosynthetic intermediates, the proposed rate-limiting step of BR biosynthesis, 22α-hydroxylation, is now known to be blocked in dwf4 mutants.
In the plant sterol biosynthetic pathway, several of the genes have been cloned or identified based on heterologous expression or sequence similarity. First, Corey et al. (1993) Proc. Natl. Acad. Sci. USA 90:11628–11632 isolated a cycloartenol synthase cDNA by heterologous complementation of yeast mutants lacking lanosterol synthase. In addition, two types of cDNAs encoding sterol methyltransferases have been isolated from soybean (Shi et al. (1996) J. Biol. Chem. 271:9384–9389) and Arabidopsis (Husselstein et al. (1996) FEBS Lett. 381:87–92). The Arabidopsis cDNA has been shown to mediate a second methyltransferase step leading to C29 sterols (Bouvier-Nave et al. (1997) Eur. J. Biochem. 246:518–529). For the 14α-demethylation reaction, Bak et al. (1997) Plant J. 11:191–201 cloned the cDNA encoding the 14-αdemethylase cytochrome P450 enzyme (CYP51) from Sorghum bicolor. Based on sequence similarity, Grebenok et al. (1997) Plant Mol. Biol. 34:891–896 identified an Arabidopsis sterol C-8 isomerase (GenBank accession number AF030357). Furthermore, an ERGOSTEROL25 (ERG25) homolog for Arabidopsis (C-4 demethylase) also has been discovered in the genome sequencing project (GenBank accession number AL021635). Finally, a sterol C-7 reductase has been cloned by heterologous expression of an Arabidopsis cDNA in yeast (Lecain et al. (1996) J. Biol. Chem. 271:10866–10873).
As compared with the wealth of cloned genes in sterol biosynthesis, only one mutant has been found in these genes. Gachotte et al. (1995) Plant J. 8:407–416 screened an ethyl methanesulfonate (EMS)-induced mutant population (22,000 M2 plants) for mutants displaying an altered sterol profile. The screen yielded one mutant, sterol1 (ste1), whose endogenous level of C-5-desaturated sterols is reduced to 30% of that of the wild type. Expression of the yeast gene ERG3 (the gene for Δ7 sterol C-5 desaturase) in the ste1-1 mutant increased the level of C-5-desaturated sterols 1.7- to 2.8-fold compared with the ste1-1 control, suggesting functional conservation of the enzymes from yeast and plants. However, visible phenotypes were not found in ste1-1 plants. Thus, the authors hypothesized that the residual 30% level of C-5-desaturated sterols was sufficient for the growth of plants.
A large collection of BR dwarf mutants have been characterized. Of the eight dwf loci identified to date, dwf3 (cpd; Szekeres et al. (1996) Cell 85:171–182), dwf4 (Choe et al. (1998) Plant Cell 10:231–243), and dwf6 (det2; Li et al. (1996) Science 272:398–401) have been shown to act in the BR biosynthetic pathway, whereas dwf2 (bri1) probably is involved in BR perception (Clouse et al. (1996) Plant Physiol. 111:671–678; Li and Chory (1997) Cell 90:929–938).